ROLE OF BILE ACIDS AND FARNESOID X RECEPTOR IN HEPATIC AUTOPHAGY AND ITS IMPLICATIONS IN ETHANOL-INDUCED HEPATOTOXICITY

Abstract

Retention of bile acids (BAs) in the liver during cholestasis plays an important role in the development of cholestatic liver injury. Several studies have reported that high concentrations of certain BAs induce cell death and inflammatory response in the liver, and BAs may promote liver tumorigenesis. Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process that regulates organelle and protein homeostasis and serves as a cell survival mechanism under a variety of stress conditions. However, it is not known if BAs modulate autophagy in hepatocytes. In the present study, we determined autophagic flux in livers of farnesoid X receptor (FXR) knockout (KO) mice that have increased concentrations of hepatic BAs and in primary cultured mouse hepatocytes that were treated with BAs. The results showed that autophagic flux was impaired in livers of FXR KO mice and in BA-treated primary mouse hepatocytes. Mechanistically, BAs did not affect the activities of cathepsin or the proteasome, but impaired autophagosomal-lysosomal fusion likely due to reduction of Rab7 protein expression and targeting to autophagosomes. In conclusion, BAs suppress autophagic flux in hepatocytes by impairing autophagosomal-lysosomal fusion, which may be implicated in bile acid-induced liver tumor promotion observed in FXR KO mice. Alcoholic liver disease encompasses a wide spectrum of pathogenesis including steatosis, fibrosis, cirrhosis, and alcoholic steatohepatitis. Acute alcohol treatment induces autophagy via FoxO3a-mediated autophagy related gene expression and protects against alcohol-induced steatosis and liver injury in mice. Moreover, inhibition of autophagy by pharmacological approach or deletion of autophagy genes exacerbates alcohol-induced steatosis and hepatotoxicity. Because we found that FXR KO mice had impaired hepatic autophagy and the role of FXR in ethanol hepatotoxicity is not known, we thus determined the hepatotoxicity and its mechanisms induced by acute ethanol treatment in FXR KO mice. In the present study, wild type and FXR KO mice were treated with acute ethanol for 16 hours. We found that ethanol treated-FXR KO mice had exacerbated hepatotoxicity and steatosis compared to wild type mice. Furthermore, we found that ethanol treatment had decreased expression of various essential autophagy genes and several other FoxO3a target genes in FXR KO mice compared with wild type mice. Mechanistically, we did not find a direct interaction between FXR and FoxO3a. Ethanol-treated FXR KO mice had increased Akt activation, increased phosphorylation of FoxO3a resulting in decreased FoxO3a nuclear retention and DNA binding. Furthermore, ethanol treatment induced hepatic mitochondrial spheroid formation in FXR KO mice, but not in wild type mice, which may serve as a compensatory alternative pathway to remove ethanol-induced damaged mitochondria in FXR KO mice. Moreover, induction of FXR with WAY-362450 protected against acute ethanol-induced steatosis, but not hepatotoxicity. These results suggest that lack of FXR impaired FoxO3a-mediated autophagy and in turn exacerbated alcohol-induced liver injury. In conclusion, this dissertation provided novel insights in how two different liver pathologies may cross talk. We demonstrated that the increased hepatic bile acid levels lead to impaired autophagy through the inhibition of autophagosomal-lysosomal fusion. Moreover, FXR deficiency exacerbated alcohol induced hepatotoxicity and steatosis likely by two mechanisms: bile acid-mediated inhibition of autophagy and Akt-mediated repression of ethanol-induced FoxO3a activation. This dissertation presented novel therapeutic targets for both cholestasic liver injury and alcoholic liver disease. Autophagy and FXR are possible therapeutic targets for cholestasis, whereas, FXR and FoxO3a emerge as novel therapeutic targets for alcoholic liver disease.